SUPPORTING INFORMATION

European Black Pine’s Phylogeography

Krassimir D. Naydenov*1, Michel K. Naydenov2, Alexander Alexandrov3, Kole Vasilevski4, Veselka Gyuleva3, Vlado Matevski5, Biljana Nikolic6, Venceslas Goudiaby7, Faruk Bogunic8, Despina Paitaridou9,Andreas Christou10,Irina Goia11, Christopher Carcaillet12, Adrian Escudero Alcantara13, Cengiz Ture14, Suleyman Gulcu15, Lorenzo Peruzzi16, Salim Kamary17,Srdjan Bojovic18, Georgi Hinkov3, Anatoly Tsarev19

TableS1Targeted cpDNA loci with sequences.

Loci
(forward) / Sequences
Pt-30204 / TATGACTGTAACTGTGATC--T--CTTTGTTCCTT------(A10-13)(G 7-15)AAGTGATCATCA
TATGACTGTAACTGTGATC--T--CTTTGTTCCTTT----(A 13)(G 9-11)AAGTGATCATCA
TATGACTGTAACTGTGATC--T--CTTTGTTCCTTTTC(A10-15)(G10-14)AAGTGATCATCA
TATGACTGTAACTGTGATCCTCCTTTGTTCCTTTTC(A 13)(G 12)AAGTGATCATCA
Pt-45002 / CTGG--TCTGATCGACCCAATATGGAACA(T12-17)ATTGATGATATTGGCAAAATCATATTTATATATCTCATG
CTGGGTCTGATCGACCCAATATGGAACA(T 16)ATTGATGATATTGGCAAAATCATATTTATATATCTCATG
Pt-71936 / AGAATC(T10-17)CAATTCCACGGATTACTCCCCATATTACC
Pt-79951 / CCAT(A11-13)GAGAATAGCTCGGCTGTATATAGCCGAGCTACAAGGATCCAGTG--AAAAAGA
CCAT(A11-13)GAGAATAGCTCGGCTGTATATAGCCGAGCTACAAGGATCCAGTGGAAAAAGA
Pt-87268 / TATTA(T10-17)CATGTATGGACCACTTGGTCCAAGTACGGGGGATATAGCTCAGTTGGTAGAGCTCCGCT--CTGC
TATTA(T 16)CATGTATGGACCACTTGGTCCAAGTACGGGGGATATAGCTCAGTTGGTAGAGCTCCGCTTCTGC
Pt-36480 / AACAAACCCTATCAAAAGTTTTATAGG(T9-13)ATGAGTGATATTGCCCCTTACTTGTCTGCTCTT
AACAAACCCTATCAAAAGTTTTATATG(T 9)ATGAGTGATATTGCCCCTTACTTGTCTGCTCTT

Table S2Protocol-algorithm for simple proportional interpolation of sequences for fragment analysis data (p.ex.Pop-X/primer pair-A/ 36 individuals)

  1. STEP-fragment analysis-electrophoretic sizing of polymerase chain reaction fragments to define electromorphs:
  2. Principe-All 36 individual from Pop-X;
  3. Result-p.ex.:two electromorphs/size variants (Size-var)–10 individuals with 140 bp and 26 individuals with 142bphave been found in Pop-X for primer pair-A;
  1. STEP- sequencing analysis (control check)
  2. Principe:12 of all individual from Pop-x have been sequenced (i.e. 33%; individual with the sequence number 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, and 34);
  3. Result-p.ex.:
  4. for Size-var with 140bp length – NON variation;
  5. for Size-var with 142bp length-YES variation: presence of two sequence variants - Seq-var (p.ex. - α and β with frequency 40% and 60% respectively);
  1. STEP-proportional interpolation (p.ex.):
  2. for 2.2.1. all individual with 140bp length have same sequencing profile (i.e. 10 individuals have same Seq-var: 140α);
  3. for 2.2.2. 40% from all individual with 142bp length have sequencing profile alpha (i.e. 10 individuals have same Seq-var: 142α); and 60% from all individual with 142bp length have sequencing profile beta (i.e. 16 individuals have same Seq-var: 142β); distribution of Seq-var: 142α and Seq-var: 142β between individuals with 142bp length is by chance.
  1. RESULT FINAL

4.1.Pop-X/primer pair-A/ 36 individuals has 3 haplotypes: 140α, 142α and 142β.

4.2.In the present algorithm the miss interpolation error is 33-50% less that publication without sequencing analysis control check;

4.3. The 142α and 142β are two distinct homoplasy haplotype (i.e. SNP variants); the 142α and 142γ are two distinct homoplasy haplotype too (i.e. SNP/SSR variants), and the 140α and 142α are two distinct haplotype (i.e. SSR variants).

………GTTTTATAGG(T10)ATGAGT……… / 140α / Pop-X
………GTTTTATAGG(T12)ATGAGT……… / 142α / Pop-X
………GTTTTATATG(T12)ATGAGT……… / 142β / Pop-X
………GTTTTATA G(T13)ATGAGT……… / 142γ / Pop-Y

Fig. S1. The haplotype group distribution by geographic region and number of individuals per haplotype. The different colors indicate that the haplotypes are: black-group 1-common for the entire natural distribution; blue-group 2- from the eastern Mediterranean sea islands-Cyprus; brown-group 3- from Morocco and western Europe i.e. the Iberian Peninsula, France and Corsica; green- group 4- from the Balkan Peninsula; grey-group 5- common to the Balkan Peninsula and Asia Minor; red- group 6- from the Apennines Peninsula (Italy); pink –group 7- from Asia Minor (Turkey); yellow-group 8- from the eastern Black sea coast.

Fig. S2.UPGMA dendrograms of 106 natural European Black Pine populations based on Rogers (1972) assuming geometric distance (at R2av = 0.94; Kalinowski 2009). The rings on the nodes indicate the bootstrap values of ≥50% on 1,000 replications. The miss classified populations are not coloured.

1

Table S3 Partitioning of genetic variance in hierrarchical analysis of AMOVA of P. nigra

Source / Percentage
of variation (PV) / F statistic
UPGMA-Rogers 1972
Among groups / 8.13 / ΦCT=0.081
Among populations within groups / 6.06 / ΦSC=0.066
Within populations / 85.82 / ΦST=0.147
“Non-admixing” model
Among groups / 7.78 / ΦCT=0.079
Among populations within groups / 7.51 / ΦSC=0.081
Within populations / 84.63 / ΦST=0.154
“Admixing” model
Among formations / 5.43 / ΦCT=0.054
Among populations within groups / 11.24 / ΦSC=0.119
Within populations / 83.33 / ΦST=0.167
Barrier-I/I
Among groups / 1.99 / ΦCT=0.020
Among populations within groups / 12.30 / ΦSC=0.125
Within populations / 85.71 / ΦST=0.145
Barrier-II/III
Among groups / 0.31 / ΦCT=0.003
Among populations within groups / 12.76 / ΦSC=0.128
Within populations / 86.93 / ΦST=0.131
Barrier-II/IV
Among groups / 2.02 / ΦCT=0.020
Among populations within groups / 11.96 / ΦSC=0.122
Within populations / 86.01 / ΦST=0.142
Barrier-III/IV
Among groups / 1.78 / ΦCT=0.018
Among populations within groups / 12.00 / ΦSC=0.122
Within populations / 86.21 / ΦST=0.140
Barrier-V/V
Among groups / 0.13 / ΦCT=0.001
Among populations within groups / 12.76 / ΦSC=0.128
Within populations / 87.11 / ΦST=0.129
Barrier-VI/VI
Among groups / -0.48 / ΦCT=-0.005
Among populations within groups / 12.94 / ΦSC=0.129
Within populations / 87.54 / ΦST=0.124

Note.All analysis are performed with p <0.0001 at 1,000 permutations; Fst =0.135 (and PV =13.47%) for non-hierarchical analysis of AMOVA.

F:\Pinus-Nigra-all-cp\Pinus-Nigra-All-cp-2015\PUB-1\TGG-2\App-S-table.doc